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Off-the-shelf hepatocyte-like cells (HLCs): Characterization of cryopreserved human mesenchymal stem cell-derived HLCs
S156
Abstracts / Toxicology Letters 258S (2016) S62–S324
These results extend our previous work and support an argument that a 72 h protocol is more suitable as a standard approach for the detection of clastogenic, aneugenic and metabolically activated chemicals in the RSMN assay. http://dx.doi.org/10.1016/j.toxlet.2016.06.1593 P08-051 Off-the-shelf hepatocyte-like cells (HLCs): Characterization of cryopreserved human mesenchymal stem cell-derived HLCs M. Cipriano 1 , B. Belém 1 , J.S. Rodrigues 3 , P.E. Cruz 2 , H. Cruz 2 , N.G. Oliveira 1 , M. Castro 1 , J.M. Santos 2 , J.P. Miranda 1,∗ 1
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal 2 ECBio S.A., Rua Henrique Paiva Couceiro, N◦ 27, 2700-451 Amadora, Portugal 3 Instituto Superior Técnico (IST), Universidade de Lisboa, Av. Rovisco Pais, N◦ 1, 1049-001 Lisboa, Portugal In vitro hepatotoxicity studies mostly rely on scarce or low biotransformation capacity cellular models, such as cryopreserved human primary hepatocytes, which present low viability and metabolic activity. Stem cell-derived HLCs have been suggested as an alternative model, by their ability to maintain a hepatic-specific phenotype. Since the differentiation protocols last at least 24 days, the aim of this work was to evaluate cryopreservation as a strategy for storing competent pre-differentiated HLCs. Mesenchymal stem cells (MSCs) at D17 of hepatic differentiation (pre-HLCs) were cryopreserved/stored for 6 or 18 months. Their ability to maturate into HLCs was evaluated 10 to 14 days after thawing (D27 and D34 of differentiation). Thawed pre-HLCs presented >90% viability, adhered to collagen-coated surface revealing epithelial morphology 7 days after thawing, independently of storage time. Moreover, fully differentiated thawed-HLCs showed glycogen storage and urea and albumin production levels similar to non-cryopreserved HLCs. The presence of drug transporters OATP-C and MRP2 was also confirmed by IF. At a functional level phase I and phase II activities were compared in cryopreserved and non-cryopreserved HLCs at D34 with both presenting similar ECOD activity; whereas cryopreserved HLCs showed ∼10-fold lower CYP3A4 activity(phase I) and ∼3.5-fold higher UGTs activity(phase II) than non-cryopreserved HLCs. These results suggest that pre-HLCs are able to maturate into HLCs after cryopreservation maintaining relevant hepatocyte-like phenotype. Upon further optimization of cell culture conditions, this may be a viable strategy to obtain a long-term and ready-to-use source of HLCs as an alternative to a time-consuming differentiation protocol. http://dx.doi.org/10.1016/j.toxlet.2016.06.1594
P08-052 Pre-validation of human small intestinal tissue model system to screen drug induced gastrointestinal toxicity and wound healing S. Ayehunie 1 , Z. Stevens 1 , T. Landry 1 , S. Letasiova 2,∗ , M. Klausner 1 1 2
MatTek Corporation, Ashland, MA, USA MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Development of reliable and reproducible primary human cell based small intestinal (SMI) tissue models that recapitulate in vivo SMI tissue phenotype, structure and function are critically needed to study gastrointestinal (GI) permeation, drug toxicity and inflammation. The validity of commonly used Caco-2 cell models is questionable due to a lack of physiological relevance and animal models often fail to predict human responses. This study describes development of 3D SMI models from primary human SMI epithelial cells and fibroblasts. Long term culture of the models and application for drug toxicity and drug permeation studies are demonstrated. The utility of the reconstructed SMI tissue models for GI drug toxicity studies was evaluated using the GI toxicant drug, indomethacin. Outcome measurements include TEER, histology and apical protein washes (sloughed epithelium). Drug permeation studies were performed using 8 drugs that utilize specific transporters (Pgp, BCRP, MRP-2, etc.). Uptake or efflux transport was analyzed by LC–MS/MS. Specific findings include: (1) the SMI tissue models can be cultured for extended time (up to 28 days) with no significant change in TEER or Lucifer yellow leakage (<2%); (2) dose dependent toxicity of indomethacin was noted with respect to reduction of TEER, epithelial damage, and epithelial sloughing; and (3) functionally active drug transport (B-to-A transport with efflux ratios >2 fold) for 7 of 13 test compounds was observed. In terms of wound healing, tissues cultured in human serum completed the wound healing process at day 6 of the culture period compared to >10 days for control tissues cultured in the absence of serum. In conclusion, the newly developed SMI tissue models appear to be promising new tools for drug safety, permeation, and wound healing studies. http://dx.doi.org/10.1016/j.toxlet.2016.06.1595 P08-053 Characterization of a novel in vitro 3D skin microtissue model for efficacy and toxicity testing S. Ströbel, N. Buschmann, A. Neeladkandhan, S. Messner ∗ , J.M. Kelm InSphero AG, Schlieren, Switzerland Transwell-based 3D human full skin models are currently used as standard model for conducting skin-related safety and efficacy studies. These traditional skin equivalent models are used since decades to assess skin corrosion and irritation. However, automaton and scaleability are one of the drawbacks of the transwell technology. Such systems require large amounts of primary cells, have limited throughput compatibility and together with a rather manual manufacturing process impeding automation assay procedures which can result in inconsistent assay readouts. Here we present a spherical skin microtissue model based on primary dermal fibroblasts and keratinocytes which consists of a dermal core tissue surrounded by a coat of layered epidermal tissue. The extracellular matrix (ECM) is solely produced by the dermal
Abstracts / Toxicology Letters 258S (2016) S62–S324
These results extend our previous work and support an argument that a 72 h protocol is more suitable as a standard approach for the detection of clastogenic, aneugenic and metabolically activated chemicals in the RSMN assay. http://dx.doi.org/10.1016/j.toxlet.2016.06.1593 P08-051 Off-the-shelf hepatocyte-like cells (HLCs): Characterization of cryopreserved human mesenchymal stem cell-derived HLCs M. Cipriano 1 , B. Belém 1 , J.S. Rodrigues 3 , P.E. Cruz 2 , H. Cruz 2 , N.G. Oliveira 1 , M. Castro 1 , J.M. Santos 2 , J.P. Miranda 1,∗ 1
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal 2 ECBio S.A., Rua Henrique Paiva Couceiro, N◦ 27, 2700-451 Amadora, Portugal 3 Instituto Superior Técnico (IST), Universidade de Lisboa, Av. Rovisco Pais, N◦ 1, 1049-001 Lisboa, Portugal In vitro hepatotoxicity studies mostly rely on scarce or low biotransformation capacity cellular models, such as cryopreserved human primary hepatocytes, which present low viability and metabolic activity. Stem cell-derived HLCs have been suggested as an alternative model, by their ability to maintain a hepatic-specific phenotype. Since the differentiation protocols last at least 24 days, the aim of this work was to evaluate cryopreservation as a strategy for storing competent pre-differentiated HLCs. Mesenchymal stem cells (MSCs) at D17 of hepatic differentiation (pre-HLCs) were cryopreserved/stored for 6 or 18 months. Their ability to maturate into HLCs was evaluated 10 to 14 days after thawing (D27 and D34 of differentiation). Thawed pre-HLCs presented >90% viability, adhered to collagen-coated surface revealing epithelial morphology 7 days after thawing, independently of storage time. Moreover, fully differentiated thawed-HLCs showed glycogen storage and urea and albumin production levels similar to non-cryopreserved HLCs. The presence of drug transporters OATP-C and MRP2 was also confirmed by IF. At a functional level phase I and phase II activities were compared in cryopreserved and non-cryopreserved HLCs at D34 with both presenting similar ECOD activity; whereas cryopreserved HLCs showed ∼10-fold lower CYP3A4 activity(phase I) and ∼3.5-fold higher UGTs activity(phase II) than non-cryopreserved HLCs. These results suggest that pre-HLCs are able to maturate into HLCs after cryopreservation maintaining relevant hepatocyte-like phenotype. Upon further optimization of cell culture conditions, this may be a viable strategy to obtain a long-term and ready-to-use source of HLCs as an alternative to a time-consuming differentiation protocol. http://dx.doi.org/10.1016/j.toxlet.2016.06.1594
P08-052 Pre-validation of human small intestinal tissue model system to screen drug induced gastrointestinal toxicity and wound healing S. Ayehunie 1 , Z. Stevens 1 , T. Landry 1 , S. Letasiova 2,∗ , M. Klausner 1 1 2
MatTek Corporation, Ashland, MA, USA MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Development of reliable and reproducible primary human cell based small intestinal (SMI) tissue models that recapitulate in vivo SMI tissue phenotype, structure and function are critically needed to study gastrointestinal (GI) permeation, drug toxicity and inflammation. The validity of commonly used Caco-2 cell models is questionable due to a lack of physiological relevance and animal models often fail to predict human responses. This study describes development of 3D SMI models from primary human SMI epithelial cells and fibroblasts. Long term culture of the models and application for drug toxicity and drug permeation studies are demonstrated. The utility of the reconstructed SMI tissue models for GI drug toxicity studies was evaluated using the GI toxicant drug, indomethacin. Outcome measurements include TEER, histology and apical protein washes (sloughed epithelium). Drug permeation studies were performed using 8 drugs that utilize specific transporters (Pgp, BCRP, MRP-2, etc.). Uptake or efflux transport was analyzed by LC–MS/MS. Specific findings include: (1) the SMI tissue models can be cultured for extended time (up to 28 days) with no significant change in TEER or Lucifer yellow leakage (<2%); (2) dose dependent toxicity of indomethacin was noted with respect to reduction of TEER, epithelial damage, and epithelial sloughing; and (3) functionally active drug transport (B-to-A transport with efflux ratios >2 fold) for 7 of 13 test compounds was observed. In terms of wound healing, tissues cultured in human serum completed the wound healing process at day 6 of the culture period compared to >10 days for control tissues cultured in the absence of serum. In conclusion, the newly developed SMI tissue models appear to be promising new tools for drug safety, permeation, and wound healing studies. http://dx.doi.org/10.1016/j.toxlet.2016.06.1595 P08-053 Characterization of a novel in vitro 3D skin microtissue model for efficacy and toxicity testing S. Ströbel, N. Buschmann, A. Neeladkandhan, S. Messner ∗ , J.M. Kelm InSphero AG, Schlieren, Switzerland Transwell-based 3D human full skin models are currently used as standard model for conducting skin-related safety and efficacy studies. These traditional skin equivalent models are used since decades to assess skin corrosion and irritation. However, automaton and scaleability are one of the drawbacks of the transwell technology. Such systems require large amounts of primary cells, have limited throughput compatibility and together with a rather manual manufacturing process impeding automation assay procedures which can result in inconsistent assay readouts. Here we present a spherical skin microtissue model based on primary dermal fibroblasts and keratinocytes which consists of a dermal core tissue surrounded by a coat of layered epidermal tissue. The extracellular matrix (ECM) is solely produced by the dermal